Molecular recognition at cholinergic synapses: Acetylcholine versus choline

Abstract

Acetylcholine (ACh) released at the vertebrate nerve-muscle synapse is hydrolyzed rapidly into choline (Cho), so endplate receptors (AChRs) are exposed to high concentrations of both of these structurally-related ligands. To understand how these receptors distinguish ACh and Cho, we used single-channel electrophysiology to measure resting affinities (binding free energies) of these and other agonists in adult-type mouse AChRs having a mutation(s) at the transmitter-binding sites. The aromatic rings of αY190, αW149 and αY198 each provide ∼50% less binding energy for Cho compared to ACh. At αY198 a phenylalanine substitution had no effect, but at αY190 this substitution caused a large, agonist-independent loss in binding energy that depended on the presence of αK145. The results suggest that i) αY190 is deprotonated by αK145 to strengthen the interaction between this benzene ring and the agonist's quaternary ammonium (QA) and ii) AChRs respond strongly to ACh because an H-bond positions the QA to interact optimally with the rings, and weakly to Cho because a different H-bond tethers the ligand to misalign the QA and form weaker interactions with the aromatic groups. The results suggest that the difference in ACh versus choline binding energies is determined by different ligand positions within a fixed protein structure.

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